The development and dynamics of the phragmoplast cytoskeleton have been analyzed in living stamen hair cells of Tradescantia. Microtubules and actin microfilaments have been identified by microinjecting either carboxyfluorescein labeled brain tubulin or rhodamine phalloidin. Examination with the confocal laser scanning microscope has permitted sequential imaging of the fluorescent cytoskeletal elements in single living cells progressing through division. Phragmoplast microtubules initially emerge through the lateral coalescence of preexisting interzone microtubules. As cytokinesis progresses, these tightly clustered microtubules shorten in length and expand centrifugally toward the cell periphery. By contrast, the phragmoplast microfilaments appear to arise de novo in late anaphase in close association with the proximal surfaces of the reconstituting daughter nuclei. The microfilaments are oriented parallel to the microtubules but conspicuously do not occupy the equatorial region where microtubules interdigitate and where the cell plate vesicles aggregate and fuse. As development proceeds the microfilaments shorten in length and expand in girth, similar to microtubules, although they remain excluded from the cell plate region. In terminal phases of cell plate formation, microtubules degrade first in the central regions of the phragmoplast and later toward the edges, whereas microfilaments break down more uniformly throughout the phragmoplast. © 1993 Wiley-Liss, Inc.